Fortification of chocolate bars by solid lipid nanoparticles of ferrous sulfate based on stearic acid and fat rich in monoacylglycerol and diacylglycerol from coconut stearin: study of physicochemical properties and sensory acceptability

ABSTRACT

Iron fortification in the form of ferrous sulfate is associated with several disadvantages, particularly related to the unpleasant after-taste. In this context, ferrous sulfate must be encapsulated by forming solid lipid nanoparticles (SLNs), which can be applied to fat-based foods such as chocolate bar. Therefore, this study aimed to provide chocolate bar fortified by SLN-ferrous sulfate with good physicochemical characteristics and sensory acceptability. The process was carried out by providing fats rich in monoacylglycerol (MAG) and diacylglycerol (DAG) from coconut stearin as a lipid matrix and emulsifier, fabricating SLN-ferrous sulfate, and fortifying SLN-ferrous sulfate in the chocolate bar at 0% (control), 2.5%, 5%, and 7.5%. The results showed that the fortification significantly affected texture, color, iron content, and sensory acceptability. The addition of SLN-ferrous sulfate of 2.5% produced chocolate bar with good physicochemical properties, which were preferred by the panelists. The chocolate bar contained iron reaching 79.23 mg/kg, a small spherulitic crystal microstructure, dominated by stable β-crystals, and no fat bloom was formed. Furthermore, SLN-ferrous sulfate based on fats rich in MAG and DAG from coconut stearin was compatible with good characteristics.

KEYWORDS:

• Chocolate bar
• Fortification
• Iron
• Coconut stearin
• Solid lipid nanoparticle

Introduction

Iron is an essential mineral with an important role in forming hemoglobin and other physiological functions but the worldwide deficiency is still high. Therefore, the risk of iron deficiency needs to be reduced through fortification in various food products. Since this metal is unstable, direct fortification can cause oxidative damage and a strong iron aroma.^[1,2] Direct fortification or consumption of iron can also have adverse side effects for gastrointestinal disorders.^[3–5] Therefore, safe and protected iron is needed to meet people’s needs, encapsulated in the form of solid lipid nanoparticles (SLNs). SLN is a colloidal system consisting of solid fat, water, and surfactant, which has been used for effective encapsulation and as a delivery system for various active compounds.^[6–9]

The use of SLN provides benefits in process control, enhancing stability, increasing bioavailability, forming a stable emulsion system, and improving the delivery system for active compounds to reach the target without damage.^[10–12] Studies proved that SLN had good delivery system capabilities for medicines, nutrients, and active compounds. This system has also been used to encapsulate iron as ferrous sulfate, using saturated fat, such as stearic acid, to produce good characteristics.^[13] However, the use of stearic acid can be detrimental to health.^[14,15] An alternative combination of safer and healthier saturated fats rich in MAG and DAG from coconut stearin is needed, which contains lots of medium-chain fatty acids. Lauric acid, specifically monolaurin, can provide good health effects.^[16–18]

SLN continues to be developed for iron encapsulation using a lipid matrix in the form of fat enriched with MAG and DAG from coconut stearin, which produces the system with high entrapment efficiency and good characteristics.^[19] In this context, SLN-ferrous sulfate should be applied for fortification in various fat-based food products, including chocolate bars. However, research on the use of SLN-ferrous sulfate for fortification of chocolate bars is still limited. Chocolate bars are a food product that is liked and consumed by all levels of society, making it suitable for use as a food vehicle for iron fortification.^[20,21] Therefore, this research novelty emphasizes the use of SLN-ferrous sulfate for the fortification of chocolate bars so that chocolate bars that are rich in iron, healthy, and have good characteristics are obtained.

This study aimed to determine the percentage of SLN-ferrous sulfate based on fats rich in MAG and DAG from coconut stearin, which can be added to chocolate bars by evaluating their sensory acceptability and physicochemical properties, to obtain chocolate bars that are rich in iron and have good physicochemical characteristics and are liked by panelists.

Materials and methods

Materials

The materials used included coconut stearin, glycerol, NaOH, tert-butanol, hexane, and citric acid for the synthesis of fat rich in MAG and DAG; stearic acid, anhydrous ferrous sulfate, tween 80, and distilled water for SLN-ferrous sulfate fabrication. Meanwhile, the ingredients used in making chocolate bars were cocoa butter, cocoa powder, sugar, and SLN-ferrous sulfate.

Synthesis of fat rich in MAG and DAG

The synthesis of fat rich in MAG and DAG was carried out through glycerolysis, according to Subroto et al..^[22] Coconut stearin was put into a reactor flask, and glycerol was added at a molar ratio of 1:5 before melting at a temperature of 60–70°C. Furthermore, the ter-butanol was added in a ratio of 1:1 to the substrate, and a 3% NaOH was added as a catalyst. The reaction was conducted at 90–100°C for 4 h, and the reaction mixture was neutralized using citric acid before extracting the fat fraction with hexane. The remaining solvent was evaporated to obtain fat rich in MAG and DAG, which was used as a solid lipid matrix in SLN-ferrous sulfate fabrication.

Fabrication of SLN-ferrous sulfate

SLN-ferrous sulfate fabrication was carried out using the double emulsion and melt dispersion method in a solvent-free system, according to Subroto et al..^[19] A total of 12 g solid fat in the form of a mixture of stearic acid and MAG and DAG resulting from glycerolysis at a ratio of 80:20 were put into a beaker glass. Subsequently, the solution was heated at a temperature of 70–75°C, and 2 mL of 10% ferrous sulfate was added to the fat mixture before homogenizing at 60–90°C for 3 min. Ultrasonication was carried out at an amplitude of 45% (200W) for 3 minutes with a setting of 40 seconds on and 1 second off to form the first emulsion (W1/O). The first emulsion mixture was then added with 20% Tween 80 solution and homogenized for 10 min. The ultrasonication was conducted at 45% amplitude (200W) for 5 min with settings of 10 seconds on and 2 seconds off to form a double emulsion (W1/O/W2). The emulsion was poured into cold water (T = 5°C), and ultrasonication was carried out again at an amplitude of 45% (200W) for 5 min with settings of 10 seconds on and 2 seconds off. Subsequently, SLN-ferrous sulfate was lyophilized using a freeze dryer at −50°C for 72 h.

SLN-ferrous sulfate fortification in chocolate bars

Chocolate bars were made following Subroto et al.^[22] and Ibrahim et al..^[23] Cocoa powder (250 g), cocoa butter (450 g), and sugar (300 g) were added slowly and alternately into the conche at a temperature of 45–50°C for 4 h until all ingredients were evenly mixed into a paste. The chocolate paste was divided into four parts, and then SLN-ferrous sulfate was added with concentrations of 0% (control), 2.5%, 5%, and 7.5%, and conching was carried out for 15 min. Subsequently, the paste was tempered using the double-boiling method. In the first and second steps, the temperatures were made stable at 50°C and lowered to 32°C. Chocolate paste was poured onto a marble table and leveled using a scraper, before lowering the temperature to 27°C. The temperature was raised until 32°C, and the paste was poured into the mold and stored in the refrigerator for 24 h at 12–15°C. Furthermore, the hardened chocolate bar was released from the mold.

Determination of polydispersity index and particle size

The particle size was measured using HORIBA SZ-100 according to Sepelevs and Reineccius,^[24] and the polydispersity index (PI) was analyzed from the representation of scattering light intensity. The SLN was diluted in aquadest at the concentration of 1–100 ppm.

Determination of entrapment efficiency

Iron’s entrapment efficiency was measured by the difference between total iron in SLN and surface iron content.^[25] Surface iron content was acquired from the supernatant of SLN-ferrous sulfate. This iron content analysis was carried out on chocolate bars that had previously been digested with HNO₃. Iron content was then analyzed by a Double Beam atomic absorption spectrophotometer U-2900/2910; Hitachi.

Determination of color

The color of the sample was determined by a chromameter of Konica Minolta CM-5.^[26] Chocolate bars cut to size 2 Х 2 cm were placed in the sample holder, and then the color chromaticity was measured by a chromameter. The samples (chocolate bars) were measured for L*, a*, and b* values at three different points.

Determination of texture profile

The texture profile of the sample was measured by TAX-T2 texture analyzer.^[27] A cylindrical probe with a 25 kg load cell and 2 mm diameter was used to measure texture, including hardness and adhesiveness. The probe speed was adjusted in stages to the conditions of a pretest, a test, and a posttest for 1, 5, and 10 mm/s, respectively, and the probe was then lowered down until it penetrated 30 mm. The parameters observed included hardness, gumminess, chewiness, adhesiveness, cohesiveness, springiness, and resilience.

Determination of microstructure

The microstructure of the crystal was observed by a polarized light microscope, according to Kinta and Hartel^[28] and Basso et al..^[29] The sample was heated at 60–80°C and then cooled to 25–30°C. The sample was spread on a glass slide at 25–30°C and viewed at three different points.

Sensory evaluation

Sensory evaluation of chocolate bars was carried out using the hedonic test method, namely a test used to determine the panelists’ level of preference for samples. Testing was conducted on 20 semi-trained panelists with 5 sensory attributes that had to be assessed: taste, color, aroma, texture, and aftertaste.^[30] The rating scales used were 1–5. Samples were coded according to variations in the concentration of SLN-ferrous sulfate added, namely 427 = 0% SLN (control), 486 = 2.5% SLN, 405 = 5% SLN, and 459 = 7.5% SLN.

Determination of polymorphism

The polymorphism of the sample was determined using the instrument of X-ray diffraction according to Le Révérend et al..^[31] The chocolate bar was placed on the slide, and then the sample was measured for diffractogram by the instrument at 2θ of 3–30 deg.

Determination of crystal morphology

Crystal morphology on the sample’s surface was determined using a Scanning Electron Microscope.^[32] The sample was cut to the size of the sample holder and then coated with gold. Samples were observed at three different points.

Statistical analysis

The data was analyzed statistically by ANOVA.¬ If there was a significant difference (p < .05), it was continued with the Duncan multiple range test using PASW Statistics ver 18.0.

Results and discussion

Properties of fat rich in MAG and DAG

The properties, including acylglycerol composition and emulsion capacity of fat rich in MAG and DAG are shown in Table 1. This showed that fat rich in MAG and DAG from glycerolysis of coconut stearin had a higher MAG content than DAG and TAG. Table 1 also showed that the glycerolysis process was effective, resulting in a high MAG content. The glycerolysis method was able to produce high levels of MAG because one mole of TAG will produce 3 moles of MAG under conditions of excess glycerol.^[33,34] Fats rich in MAG and DAG have an emulsification capacity of 98%. These results indicate that this fat has a high emulsification ability. This high emulsification ability is related to the high MAG and DAG content. MAG and DAG are emulsifiers that have been widely used commercially because of their high emulsification ability and stability.^[34,35] Furthermore, fats rich in MAG and DAG from the glycerolysis of coconut stearin can be used as a solid matrix in SLN fabrication and as an emulsifier.

Table 1. Acylglycerol composition and emulsion capacity of fat rich in MAG and DAG from coconut stearin.

Characteristics	Value
Composition of acylglycerols: Monoacylglycerol (MAG) Diacylglycerol (DAG) Triacylglycerol (TAG)	85.34 ± 0.04% 8.67 ± 0.03% 5.99 ± 0.03%
Emulsion Capacity	98.00 ± 0.04%

Characteristics of SLN-Ferrous sulfate

Characteristics of SLN-Ferrous sulfate are shown in Table 2. Based on Table 2, it can be seen that the produced SLN-ferrous sulfate had varying particle sizes in the range of 217.80–3454.40 nm. This can be caused by aggregation or clumping during fabrication and storage. This agglomeration occurred due to the attraction between particles, so the particles re-formed new particles with a relatively large size.^[36] The relatively large particle size can also be related to the polydispersity index value. In this research, the SLN-ferrous sulfate had a polydispersity index of 0.85. This showed that SLNs had a particle size distribution that was less physically stable, thereby increasing the occurrence of aggregation between particles.

Table 2. Characteristics of SLN-Ferrous sulfate.

Characteristics	Value
Particle size	217.80–3454.40 nm
Polydispersity index	0.85
Entrapment efficiency (EE)	90.98%

Table 2 also showed that SLN-ferrous sulfate had an entrapment efficiency value of 90.98%. The double emulsion system used in the SLN manufacturing process can increase entrapment efficiency by up to 95%.^[37] A high entrapment value can be caused by a good interaction between the encapsulated compound and the lipid matrix used, such that the compound is well-bound to the membrane that protects it.^[19,36] Based on this entrapment efficiency, it indicated that the SLN-ferrous sulfate was capable of trapping iron and could increase the physicochemical stability of the encapsulated compound by reducing the interaction of the compound with the external environment.^[38]

Characteristics of chocolate bars fortified by SLN-ferrous sulfate

Texture Profiles: Texture profiles of chocolate bars fortified by SLN-ferrous sulfate are shown in Table 3. The hardness, gumminess, and chewiness of the chocolate bar decreased with the higher concentration of SLN-ferrous sulfate added and were significantly different (p<.05). This value decreased significantly, especially when adding 5% and 7.5% SLN-ferrous sulfate compared to the control. This can be indicated by a softer texture. This was due to the fat that made up SLN, which was a mixture of coconut stearin, which was composed mainly of medium-chain fatty acids, such as laurate, which has a lower melting point than palmitate and stearate. Fat was the main component in making SLN-ferrous sulfate, so the higher the concentration of SLN-ferrous sulfate added, the higher the fat content in the chocolate bar. Similar results were reported by Subroto et al.,^[22] who found that the addition of SLN-gallic acid reduced the texture of chocolate bars. Adding fat to chocolate bars reduced the hardness and fracturability values of the chocolate bars produced. A low hardness value caused the chocolate to have a low melting point. So, chocolate did not maintain its solid form for a long time.^[39] The texture of food ingredients can also be affected by the water content, fat content, and structural carbohydrate content, such as cellulose, starch, and protein contained in a product.^[40]

Table 3. Texture profiles of the chocolate bars fortified by SLN-ferrous sulfate.

Concentration of SLN-Ferrous sulfate	Hardness	Adhesiveness	Springiness	Cohesiveness	Gumminess	Chewiness	Resilience
0%	3789±115^a	−12.3±2.0^a	0.90±0.03^a	0.83±0.01^a	3162±117^a	2846± 24^a	0.76±0.02^a
2.5%	3441±134 ^b	−12.8±1.6^a	0.80±0.03^b	0.77±0.07^ab	2673±311^b	2190±264^b	0.67±0.06^b
5%	2076±158^c	−11.2±0.7^a	0.77±0.02^c	0.67±0.05^bc	1400±101^c	1116±36^c	0.52±0.01^c
7.5%	1044±90^d	−7.5±1.2^b	0.83±0.11^ab	0.58±0.05^c	622± 93^d	568± 28^d	0.43±0.05^d

Mean ± SD values marked with different superscript letters in the same column are statistically significantly different at the 5% level (p<0.05).

The decrease in gumminess value was also related to the hardness value. Gumminess is the result of the calculation of the hardness value multiplied by the cohesiveness value. So, changes in gumminess values will be in line with changes in hardness. Meanwhile, changes in the chewiness value were in line with the gumminess value. This was because the chewiness value resulted from calculating gumminess multiplied by springiness.^[41,42] Table 3 also shows that chocolate bars fortified by SLN-ferrous sulfate have lower cohesiveness, springiness, resilience, and adhesiveness values compared to chocolate bars in the control sample (0%). This was caused by the addition of fat from SLN-ferrous sulfate to the chocolate bar. The fat and oil in chocolate can provide a soft, smooth, and creamy texture.^[43,44] The results of the chocolate bar texture indicated that the chocolate bar with a SLN Iron concentration of 2.5% had a value that was not significantly different from the control, so it still had a good texture profile.

Color: The color of the chocolate bars fortified by SLN-ferrous sulfate can be seen in Table 4. Based on Table 4, it was known that increasing the proportion of SLN-ferrous sulfate in chocolate bars increased the L* value significantly (p<.05). A higher L* value indicated that the chocolate bar had a lighter color. Apart from that, there was an increase and decrease in the values of a* and b* significantly (p<.05). This increase in the value of a* indicated that the color of the chocolate bar tends to be reddish, especially when adding 7.5% SLN-ferrous sulfate. Meanwhile, the increase in the b* value showed that the chocolate bar tends to be yellowish, especially with the addition of 5% and 7.5% SLN-ferrous sulfate. Changes in L*, a*, and b* had implications for changes in total color difference (∆E*).^[45] The color change in chocolate bars fortified by SLN-ferrous sulfate can be caused by interactions between iron and other compounds in chocolate, such as anthocyanins, tannins, and flavonoids.^[20,46] Adawiyah and Muhandri^[47] also reported that iron fortification in the form of ferrous sulfate can produce significant color changes in food ingredients. This was due to the interaction between iron and other components found in food ingredients. Apart from that, changes in total color difference can also be caused by non-enzymatic browning reactions during processing.^[45] However, the HUE of chocolate bars was not significantly different, indicating that changes in color chromaticity did not change the identity of the final color produced, namely dark brown.

Table 4. The color of the chocolate bars was fortified by SLN-ferrous sulfate.

Concentration of SLN-ferrous sulfate	L*	a*	b*	∆E*	HUE
0%	24.15 ± 1,53^a	4.98 ± 0,66^a	3.77 ± 0,48^a	0.00 ± 0.00^a	37.17 ± 1.89^a
2.5%	24.46 ± 1,09^a	3.98 ± 0,37^a	3.29 ± 0,25^a	1.62 ± 0.02^b	37.82 ± 2.97^a
5%	25.61 ± 2,45^ab	4.89 ± 1,01^a	4.32 ± 1,47^ab	2.19 ± 0.04^c	38.13 ± 3.06^a
7.5%	28.22 ± 1,36^b	5.67 ± 0,40^b	5.59 ± 1,15^b	3.62 ± 0.03^d	38.80 ± 3.30^a

Mean ± SD values marked with different superscript letters in the same column are statistically significantly different at the 5% level (p<0.05).

Iron content

The iron content of the chocolate bars fortified by SLN-ferrous sulfate can be seen in Table 5. The fortification of SLN-ferrous sulfate increased the iron content in the chocolate bars significantly (p<.05). The iron content in the chocolate bar was in good condition because it was encapsulated in the form of SLN. Zariwala et al.^[13] reported that SLN could protect iron from the external environment and had controlled release characteristics to increase food products’ bioavailability and iron content. This increase in iron content can also be related to the encapsulation ability of SLN, where in this study, the encapsulation efficiency of SLN Iron was 90.98% (Table 2). These characteristics and high encapsulation efficiency showed that SLN could protect iron from environmental factors and was a good delivery system for iron.^[48,49]

Table 5. The iron content of chocolate bars fortified with SLN-ferrous sulfate.

Concentration of SLN-ferrous sulfate	Iron content (mg/kg)
0%	45.35 ± 0.54^a
2.5%	79.23 ± 0.86^b
5.0%	86.79 ± 0.75^c
7.5%	114.07 ± 0.95^d

Mean ± SD values marked with different superscript letters are statistically significantly different at the 5% level (p<0.05).

Microstructure

The crystal microstructure of the chocolate bars fortified by SLN-ferrous sulfate are shown in Figure 1. The crystal microstructure in the chocolate bar fortified by SLN-ferrous sulfate was similar to the control. The crystals in the chocolate bars produced were predominantly small spherulite in large numbers, dense, and varied in shape. The crystals in the chocolate bar did not form needlelike crystals. This indicated that fat bloom was not occurring. This showed that the fat used to make SLN-ferrous sulfate was compatible with the fat used in making chocolate bars. The use of emulsifiers, namely fats rich in MAG and DAG, as well as Tween 80 in SLN-ferrous sulfate fabrication for fortification, can also help prevent fat bloom in the chocolate bars produced.^[50,51] According to James and Smith,^[52] emulsifiers could prevent the formation of fat bloom in chocolate bars. Apart from that, the tempering process carried out when making chocolate bars could change crystals into form VI, which was stable and did not trigger fat bloom.^[53] The results of this study indicated that SLN-ferrous sulfate fortification did not significantly affect the crystalline microstructure of chocolate bars and still produced good crystals.

Figure 1. Microstructure of the chocolate bars fortified by SLN-ferrous sulfate.

Sensory acceptability

The sensory acceptability of the chocolate bars fortified by SLN-ferrous sulfate are shown in Figure 2. Based on Figure 2, there were no significant differences in the sensory attributes for color and texture (p>.05) in all treatments. The texture attributes were not significantly different because SLN-ferrous sulfate had a texture that was almost the same as fat in general, so it was compatible with fat-based products. There was also no significant difference in the color attribute, which can be caused by the use of SLN as a carrier material, which reduced the interaction between iron and other food ingredients, so that there was no significant difference in the color parameters at all treatments (p>.05). This can also be caused by SLN-ferrous sulfate, which had an orange-brown color, so it did not significantly affect the chocolate bar’s dark brown color.

Figure 2. Sensory acceptability of the chocolate bars fortified by SLN-ferrous sulfate.

In the aroma attribute, there was a significant difference (p<.05) for each chocolate bar that was fortified with SLN-ferrous sulfate. This could be due to the fact that iron has a strong metallic aroma, so the higher concentration of iron resulted in a decrease in the panelists’ preference for the aroma. The reaction between iron and compounds in food can also catalyzed oxidative reactions, resulting in undesirable aromas.^[54,55] Significant differences (p<.05) also occurred in preferences for taste attributes. The panelists’ high level of taste preference was found in the SLN-ferrous sulfate 2.5% fortified chocolate bar and the control. This was because the panelists still liked products with low iron concentrations, but the smell of iron started to sting at high concentrations. According to Zariwala et al.,^[13] iron fortification in food produced a metallic taste, which caused an off-flavor in the food product. This off-flavor can also be caused by catalysis of fat oxidation in the food products.

The attribute for after-taste showed significant differences (p<.05) between treatments. The after-taste that occurred when the concentration of SLN-ferrous sulfate was increased was due to the presence of a metallic taste, which caused an off-flavor in food products and an undesirable aroma due to oxidative reactions.^[55,56] The panelists’ overall acceptance of chocolate bar products also had significant differences (p<.05) between treatments. This could be due to differences in the parameters of aroma, taste, and after-taste produced in each treatment. This was caused by iron, which can catalyze the oxidation of fat, resulting in undesirable odors and colors and producing a metallic taste in products.^[57] The results of sensory acceptability analysis played a very important role in determining the best treatment, including through the principles of traditional sensory evaluation or using fuzzy logic analysis that can be used to assign distinct codes to each of the linguistic parameters.^[58] The selected treatment was then subjected to further, more in-depth study or analysis. This study showed that the SLN-ferrous sulfate at a concentration of 2.5% had the highest level of panelist preference compared to the control and other treatments, so it was chosen as the best treatment, and then subjected to further analysis, including polymorphism and surface morphology.

Polymorphism

The polymorphism of the chocolate bars fortified by SLN-ferrous sulfate 2.5% and control (0%) can be seen by the diffractogram in Figure 3. The polymorphism in the chocolate bar in the control sample (0%) had a combination of β and β’ polymorphs. The highest diffraction peak occurred at d = 4.565 Å, 2θ = 19.427, and the intensity is 165 cps. Then, another peak formed at d = 3.581 Å, 2θ = 24.846, with an intensity of 152 cps, at d = 4.236 Å and d = 3.514 Å. This indicated that β crystals were more dominant than β’ and had a stable crystal form. These results indicated that chocolate had stable crystal characteristics (crystal types V and VI).^[31,59]

Figure 3. Diffractogram of the chocolate bars fortified by SLN-Ferrous sulfate 2.5% and 0% (control) by X-ray Diffraction.

Chocolate bars fortified by SLN-ferrous sulfate 2.5% had almost the same polymorphic profile, with the highest diffraction peak value at d = 4.564 Å, 2θ = 19.435, and high intensity at 156 cps. Another high peak formed at d = 3.569 Å, 2θ = 24.93, with an intensity of 43 cps, and another peak formed at d = 4.235 Å. This indicated that the β crystal form was more dominant than β,’ and the chocolate bar crystals were stable.^[60] The polymorphism of the chocolate bar in the control sample and fortified by SLN-ferrous sulfate 2.5% showed that the polymorphic crystallization characteristics of the chocolate bar did not have a significant difference. However, the diffraction peak intensity in the chocolate bar forticated by SLN-ferrous sulfate was smaller than the control. This may be because MAG and DAG from coconut stearin as an ingredient in making SLN-ferrous sulfate had a slightly lower degree of crystallinity, so that it can reduce the degree of crystallinity in the chocolate bars. This could also be related to the chocolate bar fortified with 2.5% SLN-ferrous sulfate having a lower hardness value than the control (Table 3). The polymorphic transition is also closely related to chocolate samples having higher hardness values, reduced cohesiveness and chewiness, and a higher melting point.^[61,62]

Surface morphology

The morphology of the chocolate bar in the control sample (0%) and fortified by SLN-ferrous sulfate 2.5% can be seen in Figure 4. The crystals of the chocolate bar in the control sample (0%) and the chocolate bar fortified by SLN-ferrous sulfate 2.5% were not significantly different. This showed that the addition of SLN-ferrous sulfate to chocolate bars had good compatibility. The resulting crystals had round, varied shapes, and did not form needlelike crystals. This study showed that no fat bloom was formed on the surface of the chocolate bar in the control sample (0%), and the chocolate bar was fortified by SLN-ferrous sulfate. These results were also confirmed by microstructure, which showed that the crystals in the chocolate bars produced were predominantly by small spherulite, dense, and did not form needlelike crystals (Figure 1). According to Dahlenborg et al.,^[63] needlelike crystals showed imperfections in the crystal form and played a role in the process of fat bloom. Setting the temperature during the tempering process can prevent the formation of needlelike crystals on the surface of the chocolate bar.^[61,64]

Figure 4. Morphology of the chocolate bars control (0%) and fortified by SLN-Ferrous sulfate 2.5% by scanning electron microscope.

Conclusion

Fortification of SLN-ferrous sulfate in chocolate bars increased the iron content, the microstructure and polymorphism of the chocolate bar remained good and were liked by the panelists, but the use of SLN-ferrous sulfate must be limited to 2.5%. Chocolate bars fortified with 2.5% SLN-ferrous sulfate had a higher level of panelist preference compared to other treatments, with a good overall acceptance and high iron content. SLN-ferrous sulfate had good compatibility in chocolate bars, which was indicated by the formation of small spherulite crystals, no needlelike crystals were formed, and the crystals were a combination of β and β’ polymorphs. Therefore, the fortification of SLN-ferrous sulfate in chocolate bars could be carried out up to 2.5%, which produced chocolate bars with good physicochemical characteristics and sensory acceptability.

Acknowledgement

The authors gratefully acknowledge Universitas Padjadjaran and The Ministry of Education, Culture, Research, and Technology of the Republic of Indonesia for their support.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work is supported by Universitas Padjadjaran, Indonesia, through the scheme of Riset Kompetensi Dosen Unpad (RKDU) with grant number: 1549/UN6.3.1/PT.00/2023.